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Hello, and welcome to iBioSeminars. My name is Professor Dianne Newman,
and I am a professor at the California Institute of Technology in the divisions of Biology and Geological and Planetary Sciences.
And I am also an investigator of the Howard Hughes Medical Institute.
So in this second part of my lecture I am going to give you an example of microbial diversity at work in
environments all over the world today, where we are going to talk about
how bacteria are able to respire a toxic compound called arsenate, arsenic V,
and how the ability of these organisms to do this can affect the geochemistry of their environment in a very significant fashion.
Now the reason that we are motivated to study microbial respiration of arsenic is because arsenic is toxic,
and the activities of microorganisms are very relevant to liberating arsenic
into water supplies that can lead to skin cancers such as that shown here
on the hands of a woman from Bangladesh.
Now these lesions that you see are coming from keratosis of the skin,
but at a more molecular level we know that arsenic is extremely harmful to cells
regardless of what oxidation state it exists in.
Arsenic can exist either as arsenate, arsenic V, and in this form it is very similar to phosphate.
And because of this the cells get confused, and they take up arsenate
when they mean to be taking up phosphate, and instead of adding inorganic phosphate onto ADP to generate energy,
they will sometimes add arsenate on.
And this is unstable and rapidly hydrolyzes, and leads to an interruption in energy generation.
Now arsenic can also exist in another form commonly within the environment and within living cells,
and that is as arsenite, arsenic III. And this form is even more deleterious to cells
than arsenate because as the more reduced form, arsenite,
it is capable of reacting with reduced sulfur groups found on proteins very readily.
And when it does this it binds and distorts these proteins
and denatures them and causes them no longer to have their normal function.
Now in the United States we have strict rules on the drinking water standards
in terms of the maximum concentration of arsenic in drinking water supplies.
Last I checked it was around 10 micrograms per liter, although this number may indeed even be smaller now.
But in other parts of the world, for example in Bangladesh,
where there is really a tragically high level of arsenic in the drinking water supplies in that country,
the concentrations can go up to 1000 micrograms per liter.
And what I want to show you here is both in the United States and in Bangladesh how significantly elevated these concentrations are
in various parts of these countries.
So in the United States... what you are looking at now is a graph that was produced by the USGS several years ago,
and it is plotting regions where arsenic concentrations get quite high.
And so in areas that are red and orange we have concentrations that are exceeding the levels
that are approved for the drinking water supply.
And I am going to focus later in the talk on a part of this map in Northern California
where arsenic concentrations are very large,
and I will describe the relevance microorganisms have in terms of affecting the geochemistry of arsenic in these habitats.
All right, so now you are looking at a plot of Bangladesh,
where the arsenic concentrations in drinking water supplies are
extremely high, very elevated, and indeed in many locations
well above what would be considered the safe level by EPA guidelines within the United States.
And a dramatic illustration of this is simply when you are looking at this plot here.
In the regions in purple the probability where the concentration of arsenic in the drinking water
is greater than 50 micrograms per liter is over 70%.
It is at least 70% in these regions, and this is a non-insignificant area of the country.
So how does arsenic get into the drinking water in these environments, and what is the connection to microbial activity?
The connection is driven by microbial metabolism and specifically anaerobic metabolisms
that occur in the sediments once oxygen is depleted from these environments.
What I am showing you here now is a very simplified view of the geochemical cycle of arsenic in a freshwater environment.
What I want to focus on is this part of the slide here
where you see this box that is depicting the transformation between arsenate, arsenic V, and arsenite.
Now these are the two primary forms of inorganic arsenic.
Other parts of this diagram that I am not going to describe
now further depict pathways that are involved in the methylation of arsenic species,
and this is an interesting subject in itself, but let's restrict our attention here now
to this inorganic cycle because this is likely to be the most important part in controlling arsenic geochemistry
in potable water supplies in many environments, such as in Bangladesh.
What we know is that in these environments
arsenic V is able to be trapped often times in association with iron minerals
where it comes down into the sediments.
And it stays there bound up to these ferric oxyhydroxide species, where it is either co-precipitating
or adsorbed onto these iron minerals,
and this arsenate because of its anionic charge likes to stick to these positively charged minerals
very well. So it remains associated with them.
And down in the sediments it will stay quite happily
if it weren't for the deposition of an electron donor, an organic carbon source, oftentimes fed by latrines
for example in Bangladesh, that provides a fertile substrate to promote microbial growth.
Now in these sediments, of course, there are other electron acceptors that can be used for respiration
and these include oxygen as a primary example, sometimes nitrate, sometimes other substrates,
but many occasions arise where these other electron acceptors that have a higher redox potential are depleted.
And once this happens, there still is an abundance of organic carbon that needs to be oxidized
and that is burned by utilizing arsenic and iron in these sediments as electron acceptors for microbial respiration.
Now when bacteria begin to respire arsenic, they reduce it from arsenic V to arsenic III,
so again that is arsenate going to arsenite.
And arsenite is uncharged at the pH of natural waters,
and as such it doesn't adsorb as well under most conditions, although it still can adsorb.
It would be too simplistic to say that it just becomes purely soluble,
but by and large I think it is fair to say that when bacteria reduce arsenate to arsenite,
this does affect its mobility, allowing arsenite to enter into the water supply, leave the sediments,
and be carried there in groundwater systems into drinking water supplies.
So knowing that arsenic respiring microorganisms are catalyzing this crucial step,
it became of interest to be able to predict their activity.
And yet, many years ago, well, not that many years ago, about 10 years ago
this was very difficult to do because there was nothing known about
the molecular mechanism underpinning microbial respiration of arsenic.
All that was known was that there were diverse organisms from the tree of life.
So if you remember from my first lecture that big phylogenetic tree I showed you,
here now is a reduced version of that where most of these organisms
you see belong to the bacterial family, although there are a few here in the archaea,
but all of these organisms are ones that interact in some fashion with arsenic.
Yet, going back ten years ago, we didn't know, as I said, how they did this.
What was the molecular biology underlying how they were able to utilize arsenic in respiration?
So my group set out to answer this question. And what we needed at the time
was a strain that we could utilize as a model system
in order to gain insight into this process.
And so we went to an environment where we thought we could isolate an arsenate respiring organism
that would be easy to work with in the lab.
And by easy to work with I mean one that we could grow on the bench,
under regular atmospheres, where we didn't have to worry about keeping it strictly anaerobic all of the time,
which up until the isolation of this organism, had been the case for the previously described arsenate respiring bacteria,
these organisms that were only able to grow under anaerobic conditions.
Now this bacterium came from a pier in Woods Hole.
And this is Woods Hole, Massachusetts. It is where the Marine Biological Laboratory is.
And a student in the microbial diversity summer course for her independent project helped me isolate an organism
from wooden chips from this pier. And the reason that we went to this wooden pier is that arsenic historically was used as a wood preservative.
And so we reasoned that if we could find bacteria attached to this wood here
they would be likely organisms that would be able to resist arsenic, because this wood was full of arsenic,
and also able to tolerate being exposed to the atmosphere, because we were going to collect
them right at the air/water interface where they would be seeing a lot of oxygen
or at least so we thought or so we hoped.
And our first enrichment was one where we put them under anaerobic conditions
in a bottle where we gave them a food source and arsenate as their sole electron acceptor for respiration.
And we asked if they could grow and reduce arsenic.
And the way we could tell if they were reducing arsenic was by whether or not the bottle was turning yellow.
And this is a very simple test. It's a test for the production of arsenic trisulfide, As2S3,
that is the molecular formula for arsenic trisulfide,
which is a yellow mineral that is produced only when arsenate is reduced to arsenite,
and then arsenite combines with sulfide to generate this yellow mineral
under the pH conditions relevant for the bottle.
So seeing this we were able then to tease out the organism catalyzing this reaction,
and we did a very targeted isolation where we required that
this organism was also capable of growing in a rich medium on a plate on the bench.
And by going back and forth between the anaerobic bottle and the rich plate, we were able to
ultimately isolate a bacterium, shown here,
that we called Shewanella sp. strain ANA-3. And the reason we named it Shewanella
is because when we sequenced its ribosomal DNA, in particular the 16S subunit,
which I mentioned before is used oftentimes, is used many, many times, in microbial phylogeny
to understand the evolutionary relatedness of an organism to other organisms.
It became quite clear that this strain that we isolated grouped within a larger genus called Shewanella.
Now, ANA-3 was a fantastic find for us because, as I told you, we demanded that it was able to grow easily on plates,
and this opened up the possibility of performing experiments
where we could do genetic manipulations and select for single colonies
on these plates like the ones you see here that had a specific mutation.
Before we entered into the whole prospect of doing mutagenesis
and genetically manipulating these organisms,
however, we wanted to verify that this organism actually could respire arsenate.
And the more rigorous way of doing this beyond just seeing whether the bottle turned yellow
was to measure the coupling of the reduction of arsenate to arsenite
to the oxidation of lactate to acetate.
Now I am only going to show you the data of arsenate reduction,
but you can take my word for it that the data also indicates
a very nice stoichiometric relationship of the molar amounts of lactate oxidizing to acetate
as arsenate being reduced to arsenite
in a one to two ratio as predicted from this metabolic formula.
So here what you see is that beginning at time zero we gave these organisms ten millimolar concentrations
of arsenate, which they then reduced and concomitantly converted to arsenite
and at the same time, they grew by many generations because this is now a logarithmic plot.
And this was what we would expect from an organism that could respire on arsenate.
So we were excited because in this bottle the only electron acceptor for growth was arsenate.
Now going forward what we wanted to do was identify what the enzymatic system was that catalyzed
the conversion of arsenate to arsenite.
And so I told you in my introductory lecture that one way that organisms have of generating ATP
is by coupling the transfer of electrons from an electron donor
in this case lactate, which is being oxidized to acetate,
through the membrane that contains a respiratory chain
made up of proteins and small molecules that can not only pass electrons through the membrane, but can also concomitantly
translocate protons out of the membrane generating this gradient that can be harnessed to make ATP.
Now at the end of this chain lies what our target was,
and that is the enzyme responsible for ultimately receiving these electrons from the chain
and passing them onto arsenate, reducing it to arsenite.
This was unknown when we began.
However, before us investigators looking at different organisms
had established very beautifully that there was a separate
detoxification system in many organisms that allowed them to resist arsenic,
and this worked as summarized as shown here where in these
detoxification schemes...when these bacteria would see
arsenic V, and it would enter into the cell via some type of phosphate transport system,
they would reduce it to arsenite via a protein called ArsC,
and then this arsenite would somehow be recognized by an efflux pump
comprised of various proteins ArsA and B that would expel arsenite from the interior of the cell.
Now this was well known, and because of this we could make use of
systems where people had previously taken strains that used to be resistant to arsenic,
genetically knocked out the genes that were required to confer that resistance,
and we could then provide genes from our organism,
Shewanella ANA-3, and ask whether or not we could restore the ability of these organisms to
resist arsenic or even to respire arsenic, whether we could confer that ability upon these organisms.
And so this was a gain of function approach, where we took DNA from Shewanella ANA-3,
and we cut it up into pieces that we could clone into a vector.
And then each of these vectors we put into different
cells of our host strain that on its own was incapable, in this case when we started, of being able simply to resist
arsenic at high concentrations. And so in screening a variety of these strains
we found a clone that conferred arsenic resistance to E. coli
as you can see here. So what you are looking at now is the optical density of overnight cultures of E. coli
that contain in this case either no plasmid at all, and that is shown here with the triangles.
And as the arsenite concentration is raised greater than 0.1 millimolar,
you see they rapidly are unable to grow at all.
Whereas with a special plasmid we found with genes from Shewanella ANA-3,
they were able to resist and grow quite nicely, even at concentrations all the way up to ten millimolar.
And here we just have a control of the vector without any DNA from our ANA-3.
Now were these genes also required for the ability to respire arsenate?
So we had identified through this gain of function approach
the Ars system that Shewanella ANA-3 had because it could complement the mutant that had been deleted in its own
native system of these Ars genes, as I told you, ArsA and ArsB and ArsC
are necessary for the detoxification pathway.
So in order to answer this question whether or not this detoxification pathway was required for respiration in ANA-3,
we wanted to answer this question because we wanted to see if there was yet another pathway out there that might
be important for arsenate respiration that was completely independent of this pathway.
We generated a transposon insertion into ArsB that encodes the protein that is involved in the efflux of arsenite from the cell,
And this had the effect of simultaneously knocking out the expression of the downstream gene, ArsC,
that encodes that arsenate reductase that I told you is inside the cell
that had been previously shown to be necessary for arsenate reduction linked to detoxification.
So when we did this now- all of this genetic manipulation was done in Shewanella ANA-3-
we found that the answer was no, that this mutant that didn't have a functional ArsB or ArsC
was still capable of respiring arsenic. And here you can see its growth
in red in optical density over the course of several days,
and yet as arsenate is being reduced in the wildtype the culture is growing quite well,
but in this mutant that we made that was able to still grow and here in red
while it could reduce arsenate, it stopped, it plateaud out and wasn't ever able to grow to the same
density as the wildtype cells. So what this indicated to us was that even though there was clearly
another enzymatic system necessary for the respiration,
of arsenate, these genes involved in detoxification were still important.
And that is intuitive. As the organism is churning through more and more arsenate,
as arsenite is accumulating within the cell,
the organism needs a mechanism to get rid of it, and this arsenic detoxification
pathway then becomes relevant whether or not
the reduction is necessary is another story but the machinery to pump out the product, arsenite,
is indeed of some relevance at high concentrations.
So now the hunt was on for the genes that encoded the respiratory arsenate reductase,
having established that there was an enzymatic activity independent of the detoxification
arsenate reductase. And here we got very lucky.
So on this initial plasmid that we cloned what we found was, as I told you, all of these genes that encoded
the system necessary for detoxification,
the Ars system. But right next door were a couple of genes
and very happily for us we picked up just the very beginning end of one of these genes in our initial clone.
And we were motivated to keep sequencing because when we sequenced this portion of our clone here
we noted that the amino terminus had some homology to enzymes in the dimethylsulfoxide reductase protein family.
And this is a protein family that is responsible for
different types of anaerobic microbial respirations, enzymes that are serving as terminal electron acceptors
for substrates other than oxygen, including dimethylsulfoxide, DMSO.
So in sequencing further we found there were these two genes downstream
shown here in red, which we named arrA and arrB.
And we surmised that because these genes were right next door to the arsA, B, C genes
that they might encode the genes for the respiratory arsenate reductase.
So this is one of these moments in science
where you just get quite lucky, and indeed it turned out to be the case.
And here is the proof. So the way to demonstrate this is to knock those genes out
and then do the same experiment I mentioned before where we gave the cells only arsenic as the electron acceptor
and asked could they reduce it and could they grow.
And here the data I am showing you is the arsenate reduction data
where the wildtype in red is capable of reducing arsenate
very rapidly whereas if we knock out either the arrA or the arrB
gene there is no reduction. And what I am not showing you is that there was no growth here in these cultures either.
And then when we complemented, when we gave back the wildtype version of these genes,
we were able to restore growth and restore arsenate reduction activity.
So having found these what this suggested to us is that
the pathway could be diagrammed as shown here in terms of electron transport.
So as I have mentioned before, the oxidative phosphorylation
process depends on electron transfer from an electron donor to an electron acceptor.
In this case, that is arsenate, which comes into the cell and gets reduced to arsenite
and the coupling of electron transfer from donors such as lactate being oxidized to acetate
through enzyme complexes and small molecules such as quinones
or menaquinones ultimately to these enzymes at the tail end that are the acceptors of these electrons.
And as these electrons are passed protons are translocated,
and this generates that battery as I explained in my first talk around this membrane
that can then be harnessed through the ATP synthase to drive ATP formation and oxidative phosphorylation.
Now what I told you at the beginning was that the ArsC protein, which is the
enzyme involved in arsenic reduction
for detoxification is inside the cell.
And what we surmised from looking at the sequence data
of our arrA and arrB proteins was that they had motifs that suggested that they were likely to be
localized here in this compartment, which is called the periplasm.
And that is the space in between the outer membrane and the cytoplasmic membrane
in gram negative bacteria. Now knowing that arsenate could be pumped into the cell
through outer membrane... or porins, transporters, so not necessarily pumped,
but just taken in through these big porins. The transporters that pump it are located here in this inner membrane.
We surmised that this enzyme, if it were located here in the periplasm,
would be identifiable if we were able to put a fluorescent tag on it as a ring around the cell.
So if the enzyme were within the cell, if you did a microscopic view of the cell
everything really, effectively, would look solidly green,
whereas if that protein were just within this outer compartment, it would visualize as a ring.
And that is what we did. And here you see sure enough, as we predicted, rings being formed.
And we verified this through more classical biochemical techniques and fractionation
to demonstrate that this activity indeed was periplasmic.
Now why does any of this matter?
Okay, at this point we have identified a novel enzyme.
We know something about where it resides, but really the motivation for this project began with
trying to understand an environmental problem, and how to develop a marker for
the activity of these organisms in the environment.
And the environments that we are talking about are sediments such as those shown here
as I said exist in Bangladesh and in many other parts of the world
where arsenic is in high abundance adsorbed onto iron minerals
that collect in these sediments. Now in the beginning I told you that the state of knowledge about arsenic respiring organisms
when we began this project was that they were phylogenetically diverse.
And so this is just a little image that illustrates that concept,
and the names are not important. Just pay attention to the dots here
The point is that the dots are everywhere on this tree,
and these are organisms that themselves are metabolically versatile.
So one would never be able to predict simply by
looking for a signature, a genetic signature of a specific species whether or not arsenate respiration
were occurring because these species are capable of respiring other metabolites, so you wouldn't know necessarily
whether they were respiring arsenic in situ
and moreover close relatives of any individual species that can respire arsenic sometimes can't,
and so the species indication is not nearly good enough for tracking this activity.
Where we got very lucky was that in our model system of Shewanella ANA-3
the gene and the protein encoded by that gene that we found
catalyzed the key step in the conversion of arsenate to arsenite, namely the arrA protein together with arrB,
was remarkably well conserved over this very evolutionarily diverse group of organisms,
and that as we continued in our studies to look into the enzymology of this family of respiratory arsenate reductases
we found that they formed this nice tight group together.
And because of this we reasoned we might be able to go after this group using probes that were
specific to detecting the expression of the gene encoding this enzyme in the environment.
So to do this we took a close look at the arrA gene, and we divided it up into sections here,
and we looked at these sections for the ones that were the most diagnostic as being unique to
the enzyme family of the arsenate reductases, and not similar in these particular regions
to other enzymes that were more broadly within this DMSO reductase family because
we didn't want a marker that would pick up this family writ large.
We wanted a marker that would specifically be able to identify
this branch of the family, the arsenate reductase branch.
So we took a look at these two domains here that we called E and F,
and we designed PCR primers that could pull up a very short fragment
in a PCR reaction that we could sequence. And we hoped that this particular stretch would be one
that would only be amplified in cells that contained
the arrA enzyme, rather the gene, and then of course that gene encodes the enzyme that catalyzes the reaction.
So what you are looking at here now are controls of bacteria that have proteins in the DMSO reductase family.
This is this whole group here in blue, but do not contain the arrA gene.
And then numbers 5 through 19, rather 6 through 19
contain or belong to organisms that are arsenate respiring organisms.
And so we took the DNA from all of these different bacteria
and we did a control to make sure we could amplify just by looking for the product of primers to target the 16S ribosomal DNA gene
and happily in our negative controls we were unable to detect a product,
yet in almost all but these last two lanes here,
even if it is very faint, take my word for it,
we did indeed see the amplification of the product we were looking for.
And so for the vast majority of these bacteria, number 19 is actually an archaean, so we weren't that concerned
about this because we would infer potentially that the arsenate reductases
might have evolved along a slightly different pathway in the archaea
than in bacteria. We nonetheless appear to have a fairly robust
short sequence that we could use to track the presence of this gene or its expression.
So with this tool in hand we then wanted to go to an environment and ask
whether or not this gene was important in controlling the geochemistry at that site.
And so the first thing that we needed to establish was whether or not
this gene and the enzyme it encoded was required for arsenate to be converted in iron rich sediments.
So for example in this site here where we have arsenic adsorbed onto ferric oxyhydroxide minerals,
would we see the reduction of arsenate to arsenite in these environments
in the absence of arrA? If the answer to that was no, then if we were able to detect
the presence of the arrA protein, or in this case what we were really doing was detecting
the expression, the messenger RNA of the gene encoding that protein,
we could have a very good reason to associate any geochemical profiles in these sediments
that showed reduction with a microbiologically catalyzed process
driven by this enzyme. So, with that as the important logical first step,
we then could go and look to see whether or not those genes were indeed present
and expressed in the sediments of interest.
So, the way we got into this is we first were able to synthesize in the laboratory
sediments that mimicked those in the natural environment that I just showed you.
And I am not going to show you all of our controls, but just one representative data file here.
But what we found was very exciting, and that was,
as we had hoped, the transformation of arsenic, where now we are looking at
arsenates again going away and being transformed to arsenite, only occurred when we had functioning arrA activity,
and that was revealed to us in these experiments by looking at the ratio of RNA and DNA for the arrA sequence,
and seeing a peak of expression maximally just at the time period where arsenate reduction to arsenite was at its maximum.
And in controls where we added organisms that we genetically deleted for their ability to express this protein,
we didn't see any arsenate reduction under this condition.
And so in the absence of some chemical reductant a biologically catalyzed process was absolutely required
for arsenate transformations under these very environmentally relevant conditions.
So let's now go to a real environment
and see how this plays out.
OK. So we chose to go to the Haiwee Reservoir, which is north of the city of Los Angeles,
and it is in the southeastern part of the state of California.
Los Angeles being right around here and San Francisco up here.
And Mono Lake over near the Nevada border,
that I told you in my introductory lecture has very high concentrations of arsenic,
feeds into this ultimately through the California aqueduct. And this aqueduct is carrying very high loads of arsenic.
So these concentrations are well in excess of what is necessary to meet EPA guidelines
for the concentrations in potable water supplies that I'll remind you are 10 micrograms per liter.
Something needs to happen, and one of the ways that arsenic is reduced
in concentration in the water is by reactions with ferric chloride
that is added to the water as it is coming down the aqueduct
at the Cottonwood Treatment Facility that is several miles upstream of the Haiwee Reservoir.
Now at the Cottonwood Treatment Facility iron chloride is injected into the water.
And when it hits the water because the pH is circa neutral
iron III in this iron chloride rapidly converts into rust, ferric oxyhydroxide amorphous iron minerals, and as I've been telling you
arsenate likes to adsorb and stick onto these minerals very well.
And so arsenic gets bound up and co-precipitated with these iron oxides
and they together come down through the LA aqueduct and
wind up sedimenting out in this channel here, leading into the Haiwee Reservoir, and this is a view of the channel.
Now it was into this particular environment that students in my lab and that of
a former colleague of mine at Caltech, Professor Janet Hering went.
And here are two students, although they have both now moved on.
They are postdocs at different locations. My student Davin Malasarn and Janet's student, Kate Campbell,
both of whom are obscured in this photo, but they went into the Haiwee sediment channel
and they took these cores. And Kate was a geochemist and she and others in Janet Hering's laboratory had been studying
these sediments for years and were very familiar with the mineralogy and the aqueous geochemistry
within these sediments. They have done extensive studies.
So Kate and Davin teamed up together, and what Davin had done was to develop, in collaboration with Chad Saltikov in my lab,
this probe to look for respiratory arsenate reductase within real sediments.
So the first thing we wanted to understand was whether the geochemical
profile within this core might indicate that microbial activities were occurring.
And as I told you, in these types of sedimentary environments,
if we see arsenate being transformed to arsenite
that is a very, very powerful indication that microbial activity must be present
because in the absence of any other type of chemical reductant which in these particular sediments is not present
arsenate would otherwise be stable.
And so what Janet and former students had been able to show using
very sophisticated X-ray spectroscopic techniques that I am now going to explain very simply to you
is that as you go down through these columns that I just showed you in the previous image the arsenic speciation changes.
And so you can see that here. These are just spectral profiles of a curve you get using a fancy form of X-ray spectroscopy
called XANES for arsenite, arsenic III, and arsenate.
And now what you are looking at are the profiles at different depths in centimeters going down into that core.
And while in the very upper layers, in the first over two centimeters
worth of the sediment, most of the arsenic is lining up with the arsenic V hump.
Very rapidly as you go below two and a half centimeters you see that the peak shifts and now it is aligned with the arsenite, arsenic III.
So this was compelling geochemical evidence that some type of microbial process was likely occurring,
and therefore the icing on the cake to help put it really together
would be to demonstrate that within these sediments
this gene, arrA, was not only present, but also being expressed into messenger RNA,
which then later of course would be converted into the protein that would catalyze the transformation of arsenate to arsenite.
And so what Davin's main contribution was, together with Kate and colleagues,
was to go into these cores, extract the DNA and extract the RNA,
sequence it, and what he found that was very impressive
was that 62 to 97% of the sequences that he found
were identical in their sequence to those from known arsenate respiring organisms.
And so here we showed indeed that messenger RNA for this arrA gene, which was our robust indicator
of the ability of the organisms to respire arsenate, was not only present,
but expressed in Haiwee sediments, and because we knew that in these sediments
the expression of this gene was required for the transformation of arsenate to arsenite
we could very firmly be able to conclude that
microbial activity and microbial respiratory activity was catalyzing this process.
So to summarize, what I hope I have taught you in this lecture
is that many microorganisms respire arsenate,
and now many more are known beyond those that I showed earlier in the talk.
The list grows larger and larger every year.
But remarkably, evolution has conserved the mechanism
whereby the vast majority of these organisms are able to catalyze that activity.
And because of this, we can look to a specific gene,
the arrA gene, as a marker for this process.
And we can generate probes to detect this gene in a variety of environments,
and these probes have been optimized more recently by Chad Saltikov who was a former postdoc in my laboratory,
but now is a professor at UC Santa Cruz who has been able to take our early work
with these primers and extend it much farther into a variety of environments.
And now this is really an established way of tracing the activity of arsenate respiring organisms in the environment,
and we hope that this will be useful in a variety of countries around the world
where arsenic is a problem, where it would be of relevance to understand
if microbial activities indeed were occurring, because that might affect remediation strategies
to limit the conversion of arsenate to arsenite in those environments.
So to acknowledge as I have been doing throughout the talk, and now here by showing
their images, of course, all of these types of projects
are performed by very talented students and postdocs
in the laboratory, and I was very lucky to work with two great guys:
Chad Saltikov who was one of the first postdocs in my lab,
and he is now a professor at UC Santa Cruz in the environmental toxicology department,
and Davin Malasarn, a graduate student in biology at Caltech who is now a postdoc at UCLA.
And together with our colleagues and collaborators at Caltech, Janet Hering and her student Kate Campbell,
and a colleague in Britain, Joanne Santini, we were able to make the discoveries that I summarized in this short talk.
All of our work has been supported by a variety of agencies, and I would like to just briefly acknowledge them here.
For this particular project, the Luce Foundation and the Packard Foundation were
very important at the beginning, and HHMI later in the work, and Chad was supported through his independent
fellowship from the National Science Foundation.
Thank you, and this is the end of the second series in the iBioSeminars lecture on microbial diversity.